The invention relates to a pharmaceutical preparation based on blood coagulation factor VII as well as to a factor VII purification method.
The coagulation of blood is triggered by a series of successive reactions of various blood coagulation factors. A deficiency of blood coagulation factors prevents the formation of fibrin from fibrinogen and thus prevents wound closure; the consequence is an increased bleeding risk or hemorrhages, respectively. This is the case if there is a deficiency of vitamin K-dependent blood coagulation factors, such as factors II, VII, IX and X, which is mainly caused by an impaired function of the liver but may also be caused by an inherited deficiency of blood coagulation factors. For a substitution treatment, the corresponding blood coagulation factors are employed. In most instances, a treatment with these preparations leads to a rapid hemostasis.
Factor VII may be recovered from a biological material, such as blood, plasma or cell cultures. If blood or plasma are the starting material, it is mostly obtained together with at least one of the structurally similar factors II, IX or X in purified form. A prothrombin-complex preparation based on factors II, VII, IX and X, or a plasma fraction containing the prothrombin complex, respectively, may likewise be used as the starting material for preparing a further purified factor VII preparation.
For the treatment of patients who suffer from a factor VIII deficiency and who have developed an inhibitor directed against factor VIII, a factor VIIa preparation frequently has been suggested. Highly purified factor VIIa preparations have been described e.g. in EP 0 082 182 and by Hedner et al., (Haemostasis 19, 335-343 (1989)).
Factor VII is relatively easily activatable to factor VIIa. For instance, it has been found that factor VII zymogen is rapidly activated by a number of physiological enzymes, such as factor IXa and factor Xa (Wildgoose et al., Blood, Vol. 73, No. 7, 1989, pp. 1888-1895).
In EP 0 770 625 it has been described that with increasing complexity of the purification procedure, activation of factor VII occurs. Accordingly, the addition of blood coagulation inhibitors, such as antithrombin III/heparin or reversible inhibitors, such as benzamidine, during an affinity-chromatographic purification have been suggested to guard against the risk of a factor VIIa formation.
Likewise, the use of benzamidine protects the factor VII molecule from proteolysis during its isolation throughout the purification procedure, as described by Radcliffe et al., Journal Biological Chemistry 250, 1975, pp. 388-395.
The problem of a factor VII activation, primarily in the presence of positively charged surfaces, e.g. in case of contact with an anion exchanger material, has been described by Pedersen et al. (Biochemistry, 28, 1989, 9331-9336). It has been found that recombinant factor VII could be purified to a homogenous protein in the presence of benzamidine. In the absence of the inhibitor, recombinant factor VII was activated spontaneously. This autocatalytic activation therefore also is a problem in preparations in which not even traces of the physiological activation components are present any longer.
Inhibitors of blood coagulation as such are not desired in a pharmaceutical preparation for treating conditions caused by the deficiency of a blood coagulation factor. Physiological inhibitors, such as antithrombin III or heparin, are being added for stability purposes, i.e. to avoid the activation of the blood coagulation factors in prothrombin complex preparations. Yet, it would be desirable to provide preparations which are sufficiently stable as far as possible without the addition of such inhibitors.
The object of the invention is to provide a pharmaceutical factor VII preparation comprising a portion of activated factor VII as low as possible and a sufficient stability in the absence of inhibitors of blood coagulation. Moreover, a purification method for producing factor VII preparations is to be provided which can be performed efficiently and which is gentle on the proteins, so as to avoid the use of inhibitors, such as benzamidine.
According to the invention, this object is achieved by a preparation based on blood coagulation factor VII comprising a portion of factor VIIa of less than 5%, having a specific activity of at least 50 U/mg and having a stability in the absence of inhibitors of blood coagulation.
Stability or xe2x80x9cstablexe2x80x9d is, as known to those of ordinary skill in the blood coagulation arts, defined herein as a preparation containing blood coagulation Factor VII made in accordance with the teachings of the present invention that retains its biological activity after purification and upon standing. Biological activity is measured by activating the Factor VII and measuring its serine protease activity as described in Example 4. A stable Factor VII preparation will retain a serine protease activity of at least 50 units/mg of protein. Units are defined consistent with the Immuno AG Immunochrom FVII:C package insert which is incorporated herein by reference in its entirety.
According to the invention, it is possible for the first time to provide a highly purified factor VII preparation without using the known inhibitors of blood coagulation, in particular without the addition of antithrombin III and/or heparin, benzamidine, soybean trypsin inhibitor, phenyl-methyl-sulfonyl fluoride or EDTA. It has been shown that under the conditions described below, factor VII is not activated during a chromatographic purification procedure, even without the protection against proteolytic enzymes or activation by contact. Thus, the highly purified factor VII preparation does not necessarily contain any one of the inhibitors listed, or less than the detection limit thereof, respectively. As soon as Factor VII a specific activity of at least 50 U/mg has been obtained in the Factor VII preparation, surprisingly it could be shown that factor VII is extremely stable (even with regard to autocatalytic activation processes) whithout requiring the addition of a specific FVII activation inhibitor.
Blood coagulation Factor VII exists as a zymogan (an inactive form of enzyme) in the circulating blood. Factor VII is activated into Factor VIIa by Factor VII specific proteases such as Factors IXa and Xa. Once activated, Factor VIIa exerts a serine protease specific activity that mediates the activation of Factor X.
The stability of the highly purified factor VII is mainlyxe2x80x94though not exclusively (cf. Pedersen et al. 1989, regarding an autocatalytic activation)xe2x80x94due to the depletion of factor VII-specific proteases, among them factors IXa and Xa. Thus, it is ensured that a preparation, such as a pharmaceutical infusion preparation, based on highly purified factor VII comprising at least 50 U/mg of protein, preferably at least 100 U/mg, most preferred at least 500 U/mg of protein, in special cases even at least 1000 U/mg up to the theoretical purity of approximately 2000 U/mg, can remain in a state ready for use over an extended period of time even in the absence of inhibitors of blood coagulation, without the portion of factor VIIa in the preparation increasing beyond the allowable extent.
The preparation according to the invention has a portion of less than 5% of factor VIIa, based on the total amount of factor VII, preferably less than 3%, most preferred less than the detection limit (e.g. in the test according to Seligson et al., Haemostasis 13, 186-191, 1983).
The preparation according to the invention is, e.g., a pharmaceutical grade concentrate which can be used to produce pharmaceutical combination preparations. Such combination preparations may comprise further active substances, such as vitamin K-dependent proteins, among them one or more of blood factors II, IX, X, protein C or protein S. Thus, e.g., a prothrombin complex preparation comprising factors II, VII, IX and X may be made into a partial prothrombin complex by admixing the factor VII preparation of the invention.
Due to its negligible load of contaminants, the factor VII infusion preparation according to the invention may be provided in relatively highly concentrated form, e.g. with a concentration of from 50 to 5000 U/ml. This substantially simplifies the administration of the preparation as a bolus injection or as a short-term infusion.
The stability of the preparation may be tested in the ready for use state so as to determine whether or not it meets the stability criteria according to the invention, i.e. by incubation at room temperature for a period of at least 12 h, preferably more than 30 h. In doing so it can be determined that the preparation according to the invention still comprises less than 5% of factor VIIa.
The preparation according to the invention may also be provided in a durable commercial form, preferably as lyophilized. Upon reconstitution, the extreme stability is shown again, and even during lyophilisation/reconstitution no negative effects occur as regards the (premature) FVII activation. Further forms are deep-frozen preparations or liquid preparations which are stable for an extended storage period, optionally after the addition of stabilizers, such as carrier proteins and/or carbohydrates, preferably at 4xc2x0 C.
In any case, despite its stability properties (also as regards an autoactivation), factor VII in the preparation according to the invention is an activatable factor VII which can be activated, e.g. in vivo, without any problems and subsequently will be blood-coagulation-active according to native factor VII. Preferably, a native factor VII protein is employed, e.g. human plasmatic factor VII, or human recombinant factor VII. In the recombination of nucleic acids, also factor VII analogs may be used which, in any case, are activatable to an equal or even a higher extent (Sridhara et al., Am. J. Hematology 53, 66-71, 1996).
According to a further aspect, the present invention relates to a method of purifying factor VII from a biological material and producing the factor VII preparation according to the invention by adsorption of factor VII on a chromatographic material, fractionated elution of factor VII with a specific activity of at least 50 U/mg, whereby the elution is carried out with a buffer without the addition of inhibitors of blood coagulation, and recovery of factor VII from the eluate.